79 results on '"Weinkauf S"'
Search Results
2. Pseudo-atomic model for Hsp26 residues 63 to 214. Please be advised that the target map is not of sufficient resolution to unambiguously position backbone or side chain atoms. This model represents a likely fit.
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Muehlhofer, M., primary, Peters, C., additional, Kriehuber, T., additional, Kreuzeder, M., additional, Kazman, P., additional, Rodina, N., additional, Reif, B., additional, Haslbeck, M., additional, Weinkauf, S., additional, and Buchner, J., additional
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- 2021
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3. Heck reactions catalyzed by oxide-supported palladium – structure–activity relationships
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Wagner, M., Köhler, K., Djakovitch, L., Weinkauf, S., Hagen, V., and Muhler, M.
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- 2000
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4. Pseudo-atomic model of a 16-mer assembly of reduced recombinant human alphaA-crystallin (non domain swapped configuration)
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Peters, C., primary, Kaiser, C.J.O., additional, Weinkauf, S., additional, Zacharias, M., additional, and Buchner, J., additional
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- 2019
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5. Dense Liquid Droplets As a Step Source for the Crystallization of Lumazine Synthase
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Gliko, O, Neumaier, N, Fischer, M, Haase, I, Bacher, A, Weinkauf, S, and Vekilov, Peter G
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Life Sciences (General) - Published
- 2004
6. High resolution electron microscopy on crystals of the icosahedral lumazine synthase/riboflavin synthase complex.
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Bachmann, L., primary, Baumeister, W., additional, Ladenstein, R., additional, Schott, K., additional, Bacher, A., additional, and Weinkauf, S., additional
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- 1990
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7. Electron microscopic observations on protein crystallization: adsorption layers, aggregates and crystal defects
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Braun, N, Tack, J, Fischer, M, Bacher, A, Bachmann, L, and Weinkauf, S
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- 2000
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8. The oligomeric distribution of SecYEG is altered by SecA and translocation ligands
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Scheuring, J, Braun, N, Nothdurft, L, Stumpf, M, Veenendaal, AKJ, Kol, S, van der Does, C, Driessen, AJM, Weinkauf, S, Veenendaal, Andreas K.J., Groningen Biomolecular Sciences and Biotechnology, Host-Microbe Interactions, Molecular Microbiology, and Faculty of Science and Engineering
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SecYEG ,Protein subunit ,Proteolipids ,CATALYTIC CYCLE ,INSERTION ,Biology ,Ligands ,SEC61P COMPLEX ,translocase ,oligomerization ,Adenosine Triphosphate ,Bacterial Proteins ,Structural Biology ,Escherichia coli ,Fluorescence Resonance Energy Transfer ,Translocase ,Inner membrane ,ER MEMBRANE ,Molecular Biology ,preprotein translocation ,Adenosine Triphosphatases ,SecYEG Translocon ,SecA Proteins ,COLI PREPROTEIN TRANSLOCASE ,electron microscopy ,YIDC ,Escherichia coli Proteins ,Cell Membrane ,Membrane Proteins ,Membrane Transport Proteins ,Translocon ,Protein Transport ,Membrane protein ,Biochemistry ,MEMBRANE TRANSLOCATION ,Membrane protein complex ,SUBUNIT ,Biophysics ,biology.protein ,ATPASE ,Protein quaternary structure ,PROTEIN-CONDUCTING CHANNEL ,Dimerization ,SEC Translocation Channels - Abstract
The multimeric membrane protein complex translocase mediates the transport of preproteins across and integration of membrane proteins into the inner membrane of Escherichia coli. The translocase consists of the peripheral membrane-associated ATPase SecA and the heterotrimeric channel-forming complex consisting of SecY, SecE and SecG. We have investigated the quaternary structure of the SecYEG complex in proteoliposomes. Fluorescence resonance energy transfer demonstrates that SecYEG forms oligomers when embedded in the membrane. Freeze-fracture techniques were used to examine the oligomeric composition under non-translocating and translocating conditions. Our data show that membrane-embedded SecYEG exists in a concentration-dependent equilibrium between monomers, dimers and tetramers, and that dynamic exchange of subunits between oligomers can occur. Remarkably, the formation of dimers and tetramers in the lipid environment is stimulated significantly by membrane insertion of SecA and by the interaction with translocation ligands SecA, preprotein and ATP, suggesting that the active translocation channel consists of multiple SecYEG complexes. (c) 2005 Elsevier Ltd. All rights reserved.
- Published
- 2005
9. Stress-induced protein 1 truncation mutant (43 - 140) from Caenorhabditis elegans
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Fleckenstein, T., primary, Kastenmueller, A., additional, Stein, M.L., additional, Peters, C., additional, Daake, M., additional, Krause, M., additional, Weinfurtner, D., additional, Haslbeck, M., additional, Weinkauf, S., additional, Groll, M., additional, and Buchner, J., additional
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- 2015
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10. Stress-induced protein 1 from Caenorhabditis elegans
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Fleckenstein, T., primary, Kastenmueller, A., additional, Stein, M.L., additional, Peters, C., additional, Daake, M., additional, Krause, M., additional, Weinfurtner, D., additional, Haslbeck, M., additional, Weinkauf, S., additional, Groll, M., additional, and Buchner, J., additional
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- 2015
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11. Hsp17.7 from Deinococcus radiodurans
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Bepperling, A., primary, Alte, F., additional, Kriehuber, T., additional, Braun, N., additional, Weinkauf, S., additional, Groll, M., additional, Haslbeck, M., additional, and Buchner, J., additional
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- 2012
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12. Molecular architectures of the 24meric eye lens chaperone alphaB- crystallin elucidated by a triple hybrid approach
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Braun, N., primary, Zacharias, M., additional, Peschek, J., additional, Kastenmueller, A., additional, Zou, J., additional, Hanzlik, M., additional, Haslbeck, M., additional, Rappsilber, J., additional, Buchner, J., additional, and Weinkauf, S., additional
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- 2011
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13. Influence of particle agglomeration on the catalytic activity of carbon-supported Pt nanoparticles in CO monolayer oxidation
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Maillard, F., primary, Schreier, S., additional, Hanzlik, M., additional, Savinova, E. R., additional, Weinkauf, S., additional, and Stimming, U., additional
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- 2005
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14. Orientation of globular proteins adsorbed on solid substrates: an electron microscopic analysis
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Braun, N., primary, Tack, J., additional, Bachmann, L., additional, and Weinkauf, S., additional
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- 1996
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15. Crystal structure determination of the SecA translocation ATPase
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Hunt, J. F., primary, Weinkauf, S., additional, Henry, L., additional, Oliver, D., additional, and Deisenhofer, J., additional
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- 1996
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16. Biosynthesis of Riboflavin: Structure and mechanism of lumazine synthase
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Bacher, A., primary, Fischer, M., additional, Kis, K., additional, Kugelbrey, K., additional, Mörtl, S., additional, Scheuring, J., additional, Weinkauf, S., additional, Eberhardt, S., additional, Schmidt-Bäse, K., additional, Huber, R., additional, Ritsert, K., additional, Cushman, M., additional, and Ladenstein, R., additional
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- 1996
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17. Quantitative evaluation of heavy metal decoration on protein molecules: contrast, specificity and resolution
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Rübenkamm, E, primary, Braun, N, additional, Bachmann, L, additional, Bacher, A, additional, Brandt, J, additional, Baumeister, W, additional, and Weinkauf, S, additional
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- 1995
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18. Metal decoration of biomacromolecules and molecular assemblies: a review
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Weinkauf, S., primary and Bachmann, L., additional
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- 1992
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19. Subunit stoichiometry and three-dimensional arrangement in proteasomes from Thermoplasma acidophilum.
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Pühler, G., primary, Weinkauf, S., additional, Bachmann, L., additional, Müller, S., additional, Engel, A., additional, Hegerl, R., additional, and Baumeister, W., additional
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- 1992
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20. Influence of particle agglomeration on the catalytic activity of carbon-supported Pt nanoparticles in CO monolayer oxidation
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Maillard, F., Schreier, S., Hanzlik, M., Savinova, E. R., Weinkauf, S., and Stimming, U.
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Fuel cell electrocatalysts usually feature high noble metal contents, and these favour particle agglomeration. In this paper a variety of synthetic approaches wet chemical deposition, electrodeposition and electrodeposition on chemically preformed Pt nuclei is employed to shed light on the influence of nanoparticle agglomeration on their electrocatalytic properties. Pt loading on model glassy carbon GC support is increased systematically from 1.8 to 10.6 μg Pt cm−2and changes in the catalyst structure are followed by transmission electron microscopy. At low metal loadings ≤5.4 μg Pt cm−2 isolated single crystalline Pt nanoparticles are formed on the support surface by wet chemical deposition from H2PtCl4precursor. An increase in the metal loading results, first, in a systematic increase of the average diameter of isolated Pt nanoparticles and, second, in coalescence of nanoparticles and formation of particle agglomerates. This behaviour is in line with the previous observations on carbon-supported noble metal fuel cell electrocatalysts. The catalytic activity of PtGC electrodes is tested in CO monolayer oxidation. In agreement with the previous studies F. Maillard, M. Eikerling, O. V. Cherstiouk, S. Schreier, E. Savinova and U. Stimming, Faraday Discuss., 2004, 125, 357, we find that the reaction is strongly size sensitive, exhibiting an increase of the reaction overpotential as the particle size decreases below ca. 3 nm. At larger particle sizes the dependence levels off, the catalytic activity of particles with diameters above 3 nm approaching that of polycrystalline Pt. Meanwhile, Pt agglomerates show remarkably enhanced catalytic activity in comparison to either isolated Pt nanopraticles or polycrystalline Pt foil, catalysing CO monolayer oxidation at ca. 90 mV lower overpotential. Enhanced catalytic activity of Pt agglomerates is ascribed to high concentration of surface defects. CO stripping voltammograms from PtGC electrodes, comprising Pt agglomerates along with isolated single crystalline Pt nanoparticles from 2 to 6 nm size, feature double voltammetric peaks, the more negative corresponding to CO oxidation on Pt agglomerates, while the more positive to CO oxidation on isolated Pt nanoparticles. It is shown that CO stripping voltammetry provides a fingerprint of the particle size distribution and the extent of particle agglomeration in carbon-supported Pt catalysts.
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- 2004
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21. Biosynthesis of riboflavin. Lumazine synthase of Escherichia coli.
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Mörtl, S, Fischer, M, Richter, G, Tack, J, Weinkauf, S, and Bacher, A
- Abstract
A gene located at 443 kilobases on the Escherichia coli chromosome (subsequently designated ribE) was expressed in a recombinant E. coli strain and was shown to code for the enzyme 6, 7-dimethyl-8-ribityllumazine synthase. The recombinant enzyme was purified to homogeneity. The protein is an icosahedral capsid of 60 subunits with a mass of about 1 MDa as shown by hydrodynamic studies and by electron microscopy. In contrast to the icosahedral lumazine synthase-riboflavin synthase complex of Bacillus subtilis, the lumazine synthase of E. coli is not physically associated with another enzyme of the riboflavin pathway, and the core of the icosahedral capsid is empty. The RIB4 gene of Saccharomyces cerevisiae was also expressed to a high level (about 40% of cellular protein) in E. coli. The recombinant protein is a pentamer of 90 kDa. An insertion of 4 amino acids into helix alpha4 is likely to hinder the formation of an icosahedral capsid by the yeast protein. The kinetic properties of lumazine synthase of E. coli, B. subtilis, and S. cerevisiae are similar.
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- 1996
22. Correlation of metal decoration and topochemistry on protein surfaces
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Weinkauf, S
- Published
- 1991
23. The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.
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Strauch A, Rossa B, Köhler F, Haeussler S, Mühlhofer M, Rührnößl F, Körösy C, Bushman Y, Conradt B, Haslbeck M, Weinkauf S, and Buchner J
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- Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Molecular Chaperones genetics, Molecular Chaperones metabolism, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Heat-Shock Proteins, Small genetics, Heat-Shock Proteins, Small metabolism
- Abstract
Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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24. Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble.
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Mühlhofer M, Peters C, Kriehuber T, Kreuzeder M, Kazman P, Rodina N, Reif B, Haslbeck M, Weinkauf S, and Buchner J
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- Binding Sites genetics, Circular Dichroism, Cryoelectron Microscopy, Fluorescence Resonance Energy Transfer, Heat-Shock Proteins chemistry, Heat-Shock Proteins genetics, Heat-Shock Response, Models, Molecular, Mutation, Phosphorylation, Protein Binding, Protein Conformation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins ultrastructure, Tandem Mass Spectrometry, Temperature, Heat-Shock Proteins metabolism, Protein Multimerization, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps., (© 2021. The Author(s).)
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- 2021
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25. Imbalances in the eye lens proteome are linked to cataract formation.
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Schmid PWN, Lim NCH, Peters C, Back KC, Bourgeois B, Pirolt F, Richter B, Peschek J, Puk O, Amarie OV, Dalke C, Haslbeck M, Weinkauf S, Madl T, Graw J, and Buchner J
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- Animals, Mice, Molecular Chaperones metabolism, Proteome metabolism, Cataract metabolism, Crystallins metabolism, Lens, Crystalline metabolism, Lens, Crystalline pathology, Protein Aggregation, Pathological
- Abstract
The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, β-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.
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- 2021
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26. Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae.
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Haslbeck M, Braun N, Stromer T, Richter B, Model N, Weinkauf S, and Buchner J
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- 2020
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27. Quantifying the insertion of membrane proteins into lipid bilayer nanodiscs using a fusion protein strategy.
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Häusler E, Fredriksson K, Goba I, Peters C, Raltchev K, Sperl L, Steiner A, Weinkauf S, and Hagn F
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- Biophysical Phenomena, Cell Membrane chemistry, Cell Membrane genetics, Humans, Membrane Proteins genetics, Phospholipids chemistry, Protein Structure, Secondary, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Voltage-Dependent Anion Channel 1 chemistry, bcl-X Protein chemistry, bcl-X Protein genetics, Lipid Bilayers chemistry, Membrane Proteins chemistry, Nanostructures chemistry, Voltage-Dependent Anion Channel 1 genetics
- Abstract
A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)
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- 2020
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28. Regulation of small heat-shock proteins by hetero-oligomer formation.
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Mymrikov EV, Riedl M, Peters C, Weinkauf S, Haslbeck M, and Buchner J
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- Caco-2 Cells, HEK293 Cells, HeLa Cells, Hep G2 Cells, Humans, MCF-7 Cells, Heat-Shock Proteins, Small metabolism, Protein Multimerization
- Abstract
Small heat-shock proteins (sHsps) compose the most widespread family of molecular chaperones. The human genome encodes 10 different sHsps (HspB1-10). It has been shown that HspB1 (Hsp27), HspB5 (αB-crystallin), and HspB6 (Hsp20) can form hetero-oligomers in vivo However, the impact of hetero-oligomerization on their structure and chaperone mechanism remains enigmatic. Here, we analyzed hetero-oligomer formation in human cells and in vitro using purified proteins. Our results show that the effect of hetero-oligomer formation on the composition of the sHsp ensembles and their chaperone activities depends strongly on the respective sHsps involved. We observed that hetero-oligomer formation between HspB1 and HspB5 leads to an ensemble that is dominated by species larger than the individual homo-oligomers. In contrast, the interaction of dimeric HspB6 with either HspB1 or HspB5 oligomers shifted the ensemble toward smaller oligomers. We noted that the larger HspB1-HspB5 hetero-oligomers are less active and that HspB6 activates HspB5 by dissociation to smaller oligomer complexes. The chaperone activity of HspB1-HspB6 hetero-oligomers, however, was modulated in a substrate-specific manner, presumably due to the specific enrichment of an HspB1-HspB6 heterodimer. These heterodimeric species may allow the tuning of the chaperone properties toward specific substrates. We conclude that sHsp hetero-oligomerization exerts distinct regulatory effects depending on the sHsps involved., (© 2020 Mymrikov et al.)
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- 2020
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29. The structure and oxidation of the eye lens chaperone αA-crystallin.
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Kaiser CJO, Peters C, Schmid PWN, Stavropoulou M, Zou J, Dahiya V, Mymrikov EV, Rockel B, Asami S, Haslbeck M, Rappsilber J, Reif B, Zacharias M, Buchner J, and Weinkauf S
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- Cryoelectron Microscopy, Humans, Lens, Crystalline chemistry, Models, Molecular, Oxidation-Reduction, Protein Conformation, Protein Multimerization, Protein Unfolding, alpha-Crystallin A Chain ultrastructure, alpha-Crystallin A Chain chemistry
- Abstract
The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.
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- 2019
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30. Small heat shock proteins: Simplicity meets complexity.
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Haslbeck M, Weinkauf S, and Buchner J
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- Animals, HSP70 Heat-Shock Proteins genetics, Humans, Protein Binding, Protein Structure, Quaternary, HSP70 Heat-Shock Proteins metabolism, Protein Folding, Protein Multimerization
- Abstract
Small heat shock proteins (sHsps) are a ubiquitous and ancient family of ATP-independent molecular chaperones. A key characteristic of sHsps is that they exist in ensembles of iso-energetic oligomeric species differing in size. This property arises from a unique mode of assembly involving several parts of the subunits in a flexible manner. Current evidence suggests that smaller oligomers are more active chaperones. Thus, a shift in the equilibrium of the sHsp ensemble allows regulating the chaperone activity. Different mechanisms have been identified that reversibly change the oligomer equilibrium. The promiscuous interaction with non-native proteins generates complexes that can form aggregate-like structures from which native proteins are restored by ATP-dependent chaperones such as Hsp70 family members. In recent years, this basic paradigm has been expanded, and new roles and new cofactors, as well as variations in structure and regulation of sHsps, have emerged., (© 2018 Haslbeck et al.)
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- 2019
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31. Structure and function of α-crystallins: Traversing from in vitro to in vivo.
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Haslbeck M, Peschek J, Buchner J, and Weinkauf S
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- Amino Acid Sequence, Animals, Binding Sites, Cataract pathology, Humans, In Vitro Techniques, Lens, Crystalline ultrastructure, Molecular Sequence Data, Protein Binding, Protein Conformation, Structure-Activity Relationship, alpha-Crystallins ultrastructure, Cataract metabolism, Lens, Crystalline chemistry, Lens, Crystalline metabolism, alpha-Crystallins chemistry, alpha-Crystallins metabolism
- Abstract
Background: The two α-crystallins (αA- and αB-crystallin) are major components of our eye lenses. Their key function there is to preserve lens transparency which is a challenging task as the protein turnover in the lens is low necessitating the stability and longevity of the constituent proteins. α-Crystallins are members of the small heat shock protein family. αB-crystallin is also expressed in other cell types., Scope of the Review: The review summarizes the current concepts on the polydisperse structure of the α-crystallin oligomer and its chaperone function with a focus on the inherent complexity and highlighting gaps between in vitro and in vivo studies., Major Conclusions: Both α-crystallins protect proteins from irreversible aggregation in a promiscuous manner. In maintaining eye lens transparency, they reduce the formation of light scattering particles and balance the interactions between lens crystallins. Important for these functions is their structural dynamics and heterogeneity as well as the regulation of these processes which we are beginning to understand. However, currently, it still remains elusive to which extent the in vitro observed properties of α-crystallins reflect the highly crowded situation in the lens., General Significance: Since α-crystallins play an important role in preventing cataract in the eye lens and in the development of diverse diseases, understanding their mechanism and substrate spectra is of importance. To bridge the gap between the concepts established in vitro and the in vivo function of α-crystallins, the joining of forces between different scientific disciplines and the combination of diverse techniques in hybrid approaches are necessary. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2016
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32. The chaperone αB-crystallin uses different interfaces to capture an amorphous and an amyloid client.
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Mainz A, Peschek J, Stavropoulou M, Back KC, Bardiaux B, Asami S, Prade E, Peters C, Weinkauf S, Buchner J, and Reif B
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- Humans, Magnetic Resonance Spectroscopy, Models, Biological, Models, Molecular, Protein Binding, Protein Conformation, Amyloid metabolism, alpha-Crystallin B Chain chemistry, alpha-Crystallin B Chain metabolism
- Abstract
Small heat-shock proteins, including αB-crystallin (αB), play an important part in protein homeostasis, because their ATP-independent chaperone activity inhibits uncontrolled protein aggregation. Mechanistic details of human αB, particularly in its client-bound state, have been elusive so far, owing to the high molecular weight and the heterogeneity of these complexes. Here we provide structural insights into this highly dynamic assembly and show, by using state-of-the-art NMR spectroscopy, that the αB complex is assembled from asymmetric building blocks. Interaction studies demonstrated that the fibril-forming Alzheimer's disease Aβ1-40 peptide preferentially binds to a hydrophobic edge of the central β-sandwich of αB. In contrast, the amorphously aggregating client lysozyme is captured by the partially disordered N-terminal domain of αB. We suggest that αB uses its inherent structural plasticity to expose distinct binding interfaces and thus interact with a wide range of structurally variable clients.
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- 2015
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33. The Chaperone Activity of the Developmental Small Heat Shock Protein Sip1 Is Regulated by pH-Dependent Conformational Changes.
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Fleckenstein T, Kastenmüller A, Stein ML, Peters C, Daake M, Krause M, Weinfurtner D, Haslbeck M, Weinkauf S, Groll M, and Buchner J
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- Amino Acid Sequence, Animals, Blotting, Western, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins classification, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Cryoelectron Microscopy, Crystallography, X-Ray, Heat-Shock Proteins, Small genetics, Heat-Shock Proteins, Small metabolism, Hydrogen-Ion Concentration, Models, Molecular, Molecular Chaperones genetics, Molecular Chaperones metabolism, Molecular Sequence Data, Mutation, Phylogeny, Protein Binding, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Temperature, Caenorhabditis elegans Proteins chemistry, Heat-Shock Proteins, Small chemistry, Molecular Chaperones chemistry, Protein Conformation
- Abstract
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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34. Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain.
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Piccoli G, Onofri F, Cirnaru MD, Kaiser CJ, Jagtap P, Kastenmüller A, Pischedda F, Marte A, von Zweydorf F, Vogt A, Giesert F, Pan L, Antonucci F, Kiel C, Zhang M, Weinkauf S, Sattler M, Sala C, Matteoli M, Ueffing M, and Gloeckner CJ
- Subjects
- Animals, Cells, Cultured, Humans, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2, Mice, Mice, Inbred C57BL, Mutant Proteins chemistry, Mutant Proteins metabolism, Mutation genetics, Neuropeptides metabolism, Neurotoxins toxicity, Parkinson Disease enzymology, Parkinson Disease genetics, Parkinson Disease pathology, Protein Binding, Protein Interaction Mapping, Protein Serine-Threonine Kinases ultrastructure, Receptors for Activated C Kinase, Structure-Activity Relationship, Synapses metabolism, Neurons metabolism, Protein Interaction Domains and Motifs, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases metabolism, Synaptic Vesicles metabolism
- Abstract
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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35. Regulated structural transitions unleash the chaperone activity of αB-crystallin.
- Author
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Peschek J, Braun N, Rohrberg J, Back KC, Kriehuber T, Kastenmüller A, Weinkauf S, and Buchner J
- Subjects
- Chromatography, Gel, Cloning, Molecular, Cryoelectron Microscopy, Electrophoresis, Polyacrylamide Gel, HSP70 Heat-Shock Proteins metabolism, HeLa Cells, Humans, Image Processing, Computer-Assisted, Molecular Chaperones metabolism, Phosphorylation, Rosaniline Dyes, alpha-Crystallin B Chain metabolism, Models, Molecular, Molecular Chaperones chemistry, Protein Conformation, alpha-Crystallin B Chain chemistry
- Abstract
The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.
- Published
- 2013
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36. Alternative bacterial two-component small heat shock protein systems.
- Author
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Bepperling A, Alte F, Kriehuber T, Braun N, Weinkauf S, Groll M, Haslbeck M, and Buchner J
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins ultrastructure, Crystallography, X-Ray, Deinococcus genetics, Deinococcus metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Heat-Shock Proteins, Small genetics, Heat-Shock Proteins, Small ultrastructure, Microscopy, Electron, Transmission, Models, Molecular, Molecular Chaperones chemistry, Molecular Chaperones genetics, Molecular Chaperones metabolism, Molecular Sequence Data, Protein Folding, Protein Multimerization, Protein Structure, Quaternary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Recombinant Proteins ultrastructure, Sequence Homology, Amino Acid, Stress, Physiological, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Heat-Shock Proteins, Small chemistry, Heat-Shock Proteins, Small metabolism
- Abstract
Small heat shock proteins (sHsps) are molecular chaperones that prevent the aggregation of nonnative proteins. The sHsps investigated to date mostly form large, oligomeric complexes. The typical bacterial scenario seemed to be a two-component sHsps system of two homologous sHsps, such as the Escherichia coli sHsps IbpA and IbpB. With a view to expand our knowledge on bacterial sHsps, we analyzed the sHsp system of the bacterium Deinococcus radiodurans, which is resistant against various stress conditions. D. radiodurans encodes two sHsps, termed Hsp17.7 and Hsp20.2. Surprisingly, Hsp17.7 forms only chaperone active dimers, although its crystal structure reveals the typical α-crystallin fold. In contrast, Hsp20.2 is predominantly a 36mer that dissociates into smaller oligomeric assemblies that bind substrate proteins stably. Whereas Hsp20.2 cooperates with the ATP-dependent bacterial chaperones in their refolding, Hsp17.7 keeps substrates in a refolding-competent state by transient interactions. In summary, we show that these two sHsps are strikingly different in their quaternary structures and chaperone properties, defining a second type of bacterial two-component sHsp system.
- Published
- 2012
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37. Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach.
- Author
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Braun N, Zacharias M, Peschek J, Kastenmüller A, Zou J, Hanzlik M, Haslbeck M, Rappsilber J, Buchner J, and Weinkauf S
- Subjects
- Cross-Linking Reagents chemistry, Cryoelectron Microscopy methods, Heat-Shock Proteins metabolism, Humans, Image Processing, Computer-Assisted methods, Imaging, Three-Dimensional methods, Magnetic Resonance Spectroscopy methods, Mass Spectrometry methods, Microscopy, Electron methods, Models, Molecular, Molecular Conformation, Mutagenesis, Site-Directed, Protein Conformation, Protein Structure, Secondary, Lens, Crystalline metabolism, alpha-Crystallin B Chain chemistry
- Abstract
The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.
- Published
- 2011
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38. Vibralactone as a tool to study the activity and structure of the ClpP1P2 complex from Listeria monocytogenes.
- Author
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Zeiler E, Braun N, Böttcher T, Kastenmüller A, Weinkauf S, and Sieber SA
- Subjects
- Bacterial Proteins chemistry, Lactones chemistry, Lactones metabolism, Microscopy, Electron, Transmission, Models, Molecular, Peptide Hydrolases chemistry, Protein Binding, Protein Subunits chemistry, Protein Subunits metabolism, Bacterial Proteins metabolism, Listeria monocytogenes enzymology, Peptide Hydrolases metabolism
- Published
- 2011
- Full Text
- View/download PDF
39. Hsp12 is an intrinsically unstructured stress protein that folds upon membrane association and modulates membrane function.
- Author
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Welker S, Rudolph B, Frenzel E, Hagn F, Liebisch G, Schmitz G, Scheuring J, Kerth A, Blume A, Weinkauf S, Haslbeck M, Kessler H, and Buchner J
- Subjects
- Cell Membrane ultrastructure, Cytosol metabolism, Gene Expression Regulation, Fungal, Genotype, Heat-Shock Proteins chemistry, Heat-Shock Proteins metabolism, Heat-Shock Response, Membrane Lipids metabolism, Osmotic Pressure, Oxidative Stress, Phenotype, Protein Folding, Protein Structure, Secondary, Protein Transport, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Sequence Deletion, Stress, Physiological, Structure-Activity Relationship, Cell Membrane metabolism, Heat-Shock Proteins genetics, Membrane Fluidity, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics
- Abstract
Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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40. Ant antennae: are they sites for magnetoreception?
- Author
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de Oliveira JF, Wajnberg E, Esquivel DM, Weinkauf S, Winklhofer M, and Hanzlik M
- Subjects
- Animals, Ants radiation effects, Electromagnetic Fields, Magnetics, Mechanotransduction, Cellular radiation effects, Sense Organs radiation effects, Ants chemistry, Ants physiology, Iron analysis, Mechanotransduction, Cellular physiology, Orientation physiology, Sense Organs chemistry, Sense Organs physiology
- Abstract
Migration of the Pachycondyla marginata ant is significantly oriented at 13 degrees with respect to the geomagnetic north-south axis. On the basis of previous magnetic measurements of individual parts of the body (antennae, head, thorax and abdomen), the antennae were suggested to host a magnetoreceptor. In order to identify Fe(3+)/Fe(2+) sites in antennae tissue, we used light microscopy on Prussian/Turnbull's blue-stained tissue. Further analysis using transmission electron microscopy imaging and diffraction, combined with elemental analysis, revealed the presence of ultra-fine-grained crystals (20-100 nm) of magnetite/maghaemite (Fe(3)O(4)/gamma-Fe(2)O(3)), haematite (alpha-Fe(2)O(3)), goethite (alpha-FeOOH) besides (alumo)silicates and Fe/Ti/O compounds in different parts of the antennae, that is, in the joints between the third segment/pedicel, pedicel/scape and scape/head, respectively. The presence of (alumo)silicates and Fe/Ti/O compounds suggests that most, if not all, of the minerals in the tissue are incorporated soil particles rather than biomineralized by the ants. However, as the particles were observed within the tissue, they do not represent contamination. The amount of magnetic material associated with Johnston's organ and other joints appears to be sufficient to produce a magnetic-field-modulated mechanosensory output, which may therefore underlie the magnetic sense of the migratory ant.
- Published
- 2010
- Full Text
- View/download PDF
41. The eye lens chaperone alpha-crystallin forms defined globular assemblies.
- Author
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Peschek J, Braun N, Franzmann TM, Georgalis Y, Haslbeck M, Weinkauf S, and Buchner J
- Subjects
- Animals, Cattle, Humans, Models, Molecular, Protein Structure, Quaternary, alpha-Crystallins ultrastructure, Lens, Crystalline chemistry, alpha-Crystallins chemistry
- Abstract
Alpha-crystallins are molecular chaperones that protect vertebrate eye lens proteins from detrimental protein aggregation. alphaB-Crystallin, 1 of the 2 alpha-crystallin isoforms, is also associated with myopathies and neuropathological diseases. Despite the importance of alpha-crystallins in protein homeostasis, only little is known about their quaternary structures because of their seemingly polydisperse nature. Here, we analyzed the structures of recombinant alpha-crystallins using biophysical methods. In contrast to previous reports, we show that alphaB-crystallin assembles into defined oligomers consisting of 24 subunits. The 3-dimensional (3D) reconstruction of alphaB-crystallin by electron microscopy reveals a sphere-like structure with large openings to the interior of the protein. alphaA-Crystallin forms, in addition to complexes of 24 subunits, also smaller oligomers and large clusters consisting of individual oligomers. This propensity might explain the previously reported polydisperse nature of alpha-crystallin.
- Published
- 2009
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- View/download PDF
42. Improvement of the quality of lumazine synthase crystals by protein engineering.
- Author
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Rodríguez-Fernández L, López-Jaramillo FJ, Bacher A, Fischer M, and Weinkauf S
- Subjects
- Bacillus subtilis enzymology, Bacillus subtilis genetics, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins standards, Crystallization methods, Crystallization standards, Multienzyme Complexes chemistry, Multienzyme Complexes genetics, Mutagenesis, Site-Directed methods, Mutagenesis, Site-Directed standards, Protein Engineering methods, Riboflavin Synthase chemistry, Riboflavin Synthase genetics, Riboflavin Synthase standards, Multienzyme Complexes standards, Protein Engineering standards
- Abstract
Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 A icosahedral capsid consisting of 60 subunits and crystallizes in space group P6(3)22 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4 A resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77 A, alpha = beta = 90.0, gamma = 120 degrees , and diffracts to 1.6 A resolution.
- Published
- 2008
- Full Text
- View/download PDF
43. Structural dynamics of archaeal small heat shock proteins.
- Author
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Haslbeck M, Kastenmüller A, Buchner J, Weinkauf S, and Braun N
- Subjects
- Heat-Shock Proteins chemistry, Microscopy, Electron, Protein Conformation, Archaeal Proteins chemistry, Archaeoglobus fulgidus, HSP20 Heat-Shock Proteins chemistry, Hot Temperature
- Abstract
Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 A. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.
- Published
- 2008
- Full Text
- View/download PDF
44. Metastable liquid clusters in super- and undersaturated protein solutions.
- Author
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Gliko O, Pan W, Katsonis P, Neumaier N, Galkin O, Weinkauf S, and Vekilov PG
- Subjects
- Algorithms, Bacillus subtilis enzymology, Chemical Phenomena, Chemistry, Physical, Crystallization, Light, Models, Chemical, Monte Carlo Method, Multienzyme Complexes chemistry, Scattering, Radiation, Solutions, Proteins chemistry
- Abstract
Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the solution, have been known to form in solutions of proteins and small-molecule substances. Here, with the protein lumazine synthase as a test system, using dynamic and static light scattering and atomic force microscopy, we demonstrate submicron size clusters of dense liquid. In contrast to the macroscopic dense liquid, these clusters are metastable not only with respect to the crystals, but also with respect to the low-concentration solution: the characteristic cluster lifetime is limited to approximately 10 s, after which they decay. The cluster population is detectable only if they occupy >10(-6) of the solution volume and have a number density >105 cm-3 for 3 to 11% of the monitored time. The cluster volume fraction varies within wide limits and reaches up to 10(-3). Increasing protein concentration increases the frequency of cluster detection but does not affect the ranges of the cluster sizes, suggesting that a preferred cluster size exists. A simple Monte Carlo model with protein-like potentials reproduces the metastable clusters of dense liquid with limited lifetimes and variable sizes and suggests that the mean cluster size is determined by the kinetics of growth and decay and not by thermodynamics.
- Published
- 2007
- Full Text
- View/download PDF
45. The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC membranes.
- Author
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Afonin S, Frey A, Bayerl S, Fischer D, Wadhwani P, Weinkauf S, and Ulrich AS
- Subjects
- Cell Membrane ultrastructure, Dimyristoylphosphatidylcholine chemistry, Dimyristoylphosphatidylcholine metabolism, Gene Products, tat metabolism, Lipid Bilayers metabolism, Magnetic Resonance Spectroscopy, Microscopy, Electron, Scanning Transmission, Models, Biological, Cell Membrane chemistry, Dimyristoylphosphatidylcholine analogs & derivatives, Gene Products, tat chemistry, Lipid Bilayers chemistry, Peptides chemistry
- Abstract
Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.
- Published
- 2006
- Full Text
- View/download PDF
46. Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis.
- Author
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Meining W, Scheuring J, Fischer M, and Weinkauf S
- Subjects
- Amino Acid Substitution, Cloning, Molecular, Crystallization methods, SEC Translocation Channels, SecA Proteins, Solvents, X-Ray Diffraction, Adenosine Triphosphatases chemistry, Bacterial Proteins chemistry, Enterococcus faecalis enzymology, Membrane Transport Proteins chemistry
- Abstract
The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 A resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 A, alpha = gamma = 90.0, beta = 119.1 degrees. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.
- Published
- 2006
- Full Text
- View/download PDF
47. Determination of liposome size: a tool for protein reconstitution.
- Author
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Vojta A, Scheuring J, Neumaier N, Mirus O, Weinkauf S, and Schleiff E
- Subjects
- Light, Lipids analysis, Microscopy, Electron, Scattering, Radiation, Solubility, Liposomes chemistry, Proteins chemistry, Spectrophotometry, Atomic
- Abstract
Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological function. Many protocols exist for this procedure and for the subsequent analysis of proteins. Here, we establish a procedure for preparation and analysis of liposomes with a lipid composition reflecting the outer envelope of chloroplasts. First, the stability of the liposomes in different buffer systems was investigated to provide information for the storage of the reconstituted system. Then, the size of the liposomes created by filtration through a polycarbonate filter dependent on the lipid composition was analyzed. Subsequently, solubilization of the liposomes composed of lipids with the outer envelope composition by dodecylmaltoside and octylglucoside as a preceding step of reconstitution was studied. Finally, we developed a straightforward method to determine the size of liposomes by absorption spectroscopy. The described setup allows the construction of reconstitution protocols, including the final determination of the liposome size.
- Published
- 2005
- Full Text
- View/download PDF
48. A metastable prerequisite for the growth of lumazine synthase crystals.
- Author
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Gliko O, Neumaier N, Pan W, Haase I, Fischer M, Bacher A, Weinkauf S, and Vekilov PG
- Subjects
- Crystallization, Enzyme Stability, Kinetics, Light, Scattering, Radiation, Solutions, Thermodynamics, Multienzyme Complexes chemistry
- Abstract
Dense liquid phases, metastable with respect to a solid phase, form in solutions of proteins and small-molecule materials. They have been shown to serve as a prerequisite for the nucleation of crystals and other ordered solid phases. Here, using crystals of the protein lumazine synthase from Bacillus subtilis, which grow by the generation and spreading of layers, we demonstrate that within a range of supersaturations the only mechanism of generation of growth layers involves the association of submicrometer-size droplets of the dense liquid to the crystal surface. The dense liquid is metastable not only with respect to the crystals, but also with respect to the low-concentration solution: dynamic light scattering reveals that the droplets' lifetime is limited to several seconds, after which they decay into the low-concentration solution. The short lifetime does not allow growth to detectable dimensions so that liquid-liquid phase separation is not observed within a range of conditions broader than the one used for crystallization. If during their lifetime the droplets encounter a crystal surface, they lower their free energy not by decay, but by transformation into crystalline matter, ensuring perfect registry with the substrate. These observations illustrate two novel features of phase transformations in solutions: the existence of doubly metastable, short-lifetime dense phases and their crucial role for the growth of an ordered solid phase.
- Published
- 2005
- Full Text
- View/download PDF
49. Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae.
- Author
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Haslbeck M, Braun N, Stromer T, Richter B, Model N, Weinkauf S, and Buchner J
- Subjects
- Gene Deletion, Heat-Shock Proteins genetics, Protein Denaturation physiology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins genetics, Cytosol metabolism, Heat-Shock Proteins metabolism, Proteome metabolism, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.
- Published
- 2004
- Full Text
- View/download PDF
50. Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA.
- Author
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Hunt JF, Weinkauf S, Henry L, Fak JJ, McNicholas P, Oliver DB, and Deisenhofer J
- Subjects
- Adenosine Diphosphate chemistry, Adenosine Triphosphate chemistry, Amino Acid Motifs, Amino Acid Sequence, Bacterial Proteins metabolism, Binding Sites, Crystallization, Crystallography, X-Ray, DNA Helicases chemistry, DNA, Bacterial chemistry, DNA, Bacterial metabolism, DNA, Single-Stranded chemistry, DNA, Single-Stranded metabolism, Dimerization, Escherichia coli, Eukaryotic Initiation Factor-4A, Fluorescence Polarization, Fourier Analysis, Hydrogen Bonding, Ligands, Models, Molecular, Molecular Sequence Data, Peptide Initiation Factors chemistry, Peptides chemistry, Protein Binding, Protein Conformation, Protein Folding, Protein Precursors metabolism, Protein Structure, Secondary, SEC Translocation Channels, SecA Proteins, Temperature, Adenosine Diphosphate metabolism, Adenosine Triphosphatases chemistry, Adenosine Triphosphatases metabolism, Adenosine Triphosphate metabolism, Bacillus subtilis enzymology, Bacterial Proteins chemistry, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins metabolism, Protein Structure, Tertiary
- Abstract
The SecA adenosine triphosphatase (ATPase) mediates extrusion of the amino termini of secreted proteins from the eubacterial cytosol based on cycles of reversible binding to the SecYEG translocon. We have determined the crystal structure of SecA with and without magnesium-adenosine diphosphate bound to the high-affinity ATPase site at 3.0 and 2.7 angstrom resolution, respectively. Candidate sites for preprotein binding are located on a surface containing the SecA epitopes exposed to the periplasm upon binding to SecYEG and are thus positioned to deliver preprotein to SecYEG. Comparisons with structurally related ATPases, including superfamily I and II ATP-dependent helicases, suggest that the interaction geometry of the tandem motor domains in SecA is modulated by nucleotide binding, which is shown by fluorescence anisotropy experiments to reverse an endothermic domain-dissociation reaction hypothesized to gate binding to SecYEG.
- Published
- 2002
- Full Text
- View/download PDF
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